U.S. patent number 10,166,264 [Application Number 15/249,234] was granted by the patent office on 2019-01-01 for bacteriophage and antibacterial composition comprising the same.
This patent grant is currently assigned to CJ CHEILJEDANG CORPORATION. The grantee listed for this patent is CJ CHEILJEDANG CORPORATION. Invention is credited to Young Wook Cho, Jae Won Kim, Young Sa Kim, Eun Mi Shin, Si Yong Yang.
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United States Patent |
10,166,264 |
Yang , et al. |
January 1, 2019 |
Bacteriophage and antibacterial composition comprising the same
Abstract
The present invention relates to a bacteriophage having a
specific bactericidal activity against Salmonella, a composition
for the prevention or treatment of infectious diseases comprising
the bacteriophage as an active ingredient, an antibiotic comprising
the bacteriophage as an active ingredient, an animal feed or
drinking water comprising the bacteriophage as an active
ingredient, and a sanitizer or cleaner comprising the bacteriophage
as an active ingredient. The bacteriophage of the present invention
has a specific bactericidal activity against Salmonella
choleraesuis, Salmonella typhimurium, Salmonella derby, Salmonella
infantis or Salmonella newport with no influences on beneficial
bacteria, as well as excellent acid- and heat-resistance and
desiccation tolerance.
Inventors: |
Yang; Si Yong (Incheon,
KR), Kim; Jae Won (Seoul, KR), Cho; Young
Wook (Seoul, KR), Kim; Young Sa (Seoul,
KR), Shin; Eun Mi (Incheon, KR) |
Applicant: |
Name |
City |
State |
Country |
Type |
CJ CHEILJEDANG CORPORATION |
Seoul |
N/A |
KR |
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Assignee: |
CJ CHEILJEDANG CORPORATION
(Seoul, KR)
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Family
ID: |
47915017 |
Appl.
No.: |
15/249,234 |
Filed: |
August 26, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170128504 A1 |
May 11, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14345642 |
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9717768 |
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PCT/KR2012/007555 |
Sep 20, 2012 |
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Foreign Application Priority Data
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Sep 20, 2011 [KR] |
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10-2011-0094648 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
35/76 (20130101); C12R 1/91 (20130101); A23K
50/30 (20160501); A23K 20/147 (20160501); A01N
63/00 (20130101); A23K 50/10 (20160501); A61P
31/04 (20180101); A23K 20/10 (20160501); C12N
7/00 (20130101); A23K 50/75 (20160501); A23K
20/195 (20160501); C12N 2795/10321 (20130101); C12N
2795/10331 (20130101); C12N 2795/10332 (20130101) |
Current International
Class: |
A61K
35/76 (20150101); A01N 63/00 (20060101); C12N
7/00 (20060101); A23K 50/10 (20160101); A23K
50/30 (20160101); A23K 50/75 (20160101); A23K
20/147 (20160101); A23K 20/10 (20160101); A23K
20/195 (20160101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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10-2009-0030386 |
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Mar 2009 |
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KR |
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10-2010-0075263 |
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Jul 2010 |
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KR |
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1020140015896 |
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Apr 2016 |
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KR |
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2010-074433 |
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Jul 2010 |
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WO |
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Other References
English MT.Kyoung, W.L. 2016.Fermented extract of Gastrodia elata
with excellent antioxidant activity and taste and manufacturing
method thereof. Korean Patent Application No. 1020140015896. Pub.
Date: Apr. 5, 2016. pp. 1-26. specif. p. 8, para. 5. cited by
examiner .
Ackermann, H.-W., "Frequency of morphological phage descriptions in
the year 2000," Arch Virol 146: 843-857, 2001. cited by applicant
.
Nnalue et al., "Lysogenization of Salmonella choleraesuis by phage
14 increases average length of O-antigen chains, serum resistance
and intraperitoneal mouse virulence," Microbial Pathogenesis 8:
393-402, 1990. cited by applicant .
O'Flynn et al., "The newly isolated lytic bacteriophages st104a and
st104b are highly virulent against Salmonella enterica," Journal of
Applied Microbiology 101:251-259, 2006. cited by applicant.
|
Primary Examiner: Claytor; Renee
Assistant Examiner: Papciak; Sharon M.
Attorney, Agent or Firm: Cooley LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No.
14/345,642, filed Mar. 18, 2014, which is a U.S. national phase
application of International PCT Patent Application No.
PCT/KR2012/007555, filed Sep. 20, 2012, which claims priority to
Korean Patent Application No. 10-2011-0094648, filed Sep. 20, 2011.
The foregoing applications are incorporated herein by reference in
their entireties.
Claims
The invention claimed is:
1. A method of preparing an animal feed composition, the method
comprising: providing a bacteriophage composition comprising
bacteriophage as an active ingredient and a additive; and mixing
the composition with an animal feed base to provide the animal feed
composition, wherein the bacteriophage has nucleic acid sequences
of SEQ ID NOs: 1 to 4.
2. The method of preparing an animal feed composition according to
claim 1, wherein the bacteriophage is bacteriophage .PHI.CJ11
deposited under accession number KCCM11208P.
3. The method of preparing an animal feed composition according to
claim 1, wherein the bacteriophage composition further comprises
other non-pathogenic microorganisms.
4. The method of preparing an animal feed composition according to
claim 3, wherein the other non-pathogenic microorganisms are
selected from the group consisting of Bacillus subtilis,
Lactobacillus sp. strain, filamentous fungi, and yeast including
Saccharomyces cerevisiae.
5. The method of claim 1, wherein the bacteriophage is in an amount
of 0.05 to 10 by dry weight based on 100 parts by dry weight of the
feed composition.
6. A method of feeding an animal feed composition to an animal, the
method comprising: preparing the animal feed composition according
to the method of claim 1; and feeding the animal feed composition
as a feed to the animal.
7. A method of preparing a drinking water composition, the method
comprising: providing a bacteriophage composition comprising
bacteriophage as an active ingredient and a additive; and mixing
the composition with drinking water to provide the drinking water
composition, wherein the bacteriophage has nucleic acid sequences
of SEQ ID NOs: 1 to 4.
8. The method of preparing a drinking water composition according
to claim 7, wherein the bacteriophage is bacteriophage .PHI.CJ11
deposited under accession number KCCM11208P.
9. The method of preparing a drinking water composition according
to claim 7, wherein the bacteriophage composition further comprises
other non-pathogenic microorganisms.
10. The method of preparing a drinking water composition according
to claim 9, wherein the other non-pathogenic microorganisms are
selected from the group consisting of Bacillus subtilis,
Lactobacillus sp. strain, filamentous fungi, and yeast including
Saccharomyces cerevisiae.
11. The method of claim 7, wherein the bacteriophage is in an
amount of 0.0001 to 0.01 by dry weight based on 100 parts by dry
weight of the drinking water.
12. A method of feeding a drinking water composition to an animal,
the method comprising: preparing the drinking water composition
according to the method of claim 7; and feeding the drinking water
composition as a feed to the animal.
Description
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided
in text format in lieu of a paper copy, and is hereby incorporated
by reference into the specification. The name of the text file
containing the Sequence Listing is HANO_025_01US_ST25.txt. The text
file is 11 KB, was created on Aug. 26, 2016, and is being submitted
electronically via EFS-Web.
TECHNICAL FIELD
The present invention relates to a novel bacteriophage and an
antibacterial composition comprising the same.
BACKGROUND ART
Salmonella has an average genomic GC content of 50-52%, which is
similar to that of Escherichia coli and Shigella. The genus
Salmonella is a pathogenic microorganism that causes infections in
livestock as well as in humans. Serological division has it that
Salmonella enterica, a species of Salmonella bacterium, has a
variety of serovars including Salmonella gallinarum, Salmonella
pullorum, Salmonella typhimurium (ST), Salmonella enteritidis (SE),
Salmonella typhi, Salmonella choleraesuis (SC), Salmonella derby
(SD). Of them, choleraesuis and derby are swine-adapted pathogens,
gallinarum and pullorum are fowl-adapted pathogens, typhimurium and
enteritis are pathogenic for humans and animals, and typhi is a
human-adapted pathogen, all of which cause illness in their
respective species, resulting in tremendous damage to farmers and
consumers (Zoobises Report; United Kingdom 2003).
Recently, implementation of HACCP (Hazard analysis and critical
control points) has become a mandatory requirement for all
slaughterhouses in Korea as of Jul. 1, 2003, because of the high
risk of contamination in the course of manufacturing livestock
products with salmonella, which causes direct damage to pigs, and
meat hygiene and is found in the digestive tract of pigs (Jae-gil
Yeh. Characterization and Counterplan of Salmonellosis in Pigs.
Monthly Magazine of Pig Husbandry. 2004).
Paratyphoid, the acute or chronic infectious disease in the
digestive tract of pig caused by salmonella infection, is
characterized by gastroenteritis and septicaemia and mainly occurs
during the fatting period. In particular, some of pathogenic
bacteria that cause this disease can cause food poisoning in humans
through meat ingestion, and thus it is a disease having major
public health importance. A variety of types of salmonella bacteria
can be pathogenic. Among them, Salmonella choleraesuis and
Salmonella typhisuis known to cause hog cholera are the major
causes of acute salmonella septicaemia. Acute enteritis occurs
during the fattening period, and is accompanied by irregular
appetite, severe watery diarrhea, high fever, loss of vitality,
pneumonia, and nervous signs. Discoloration of the skin may occur
in some severe cases. Salmonella typhimurium, Salmonella
enteritidis, and Salmonella derby are the major causes of chronic
enteritis.
Salmonellosis is caused by oral route through feed or water
contaminated with salmonella, and thus these routes should be
prevented. Contaminated feed, raw materials or water, or adult pigs
carrying the pathogen can be major sources of infection. During the
acute period of infection, pigs shed up to 10.sup.6 Salmonella
choleraesuis or 10.sup.7 Salmonella typhimurium per gram of feces.
However, many experimental infections reported successful disease
reproduction with a dose of 10.sup.8 to 10.sup.11 Salmonella. In an
experiment injecting 10.sup.3 Salmonella into pigs, the injected
pigs showed no symptoms of the disease, but other pigs raised in
the same pen showed typical clinical symptoms. These results
indicate that a large amount of salmonella grow in naturally
infected pigs, resulting in the infection of other pigs (Jung-Bok
Lee. Control of the Recent Outbreaks of Porcine Salmonella and
Proliferative Enteropathy. Korea Swine Association. 2009).
At present, severe viral infections such as Porcine Reproductive
and Respiratory Syndrome (PRRS) and Porcine Circovirus (PCV2) have
been causing tremendous economic losses to the swine industry in
Korea, and thus disease management has been focused on these
diseases. Since these bacterial diseases may cause tremendous
damage comparable to that caused by viral diseases beginning with a
ban on the use of in-feed antibiotics and an investigation of
disease occurrence or disease management should be performed in
advance (Jung-Bok Lee. Control of the Recent Outbreaks of Porcine
Salmonella and Proliferative Enteropathy, Infectious Disease
Laboratory, College of Veterinary Medicine, Konkuk University,
Livestock Product Safety, 2010) (Robert W. Wills, Veterinary
Microbiology, 1999). Meanwhile, bacteriophage, also called phage,
is a specialized type of virus that infects only particular
bacteria and controls the growth of bacteria, and can
self-replicate only inside the host bacteria. After the discovery
of bacteriophages, a great deal of faith was initially placed in
their use for infectious-disease therapy. However, when broad
spectrum antibiotics came into common use, bacteriophages were seen
as unnecessary due to a specific target spectrum. Antibiotics or
antimicrobial agents have been widely used for the treatment of
infectious diseases caused by bacterial infection. Nevertheless,
the misuse and overuse of antibiotics resulted in rising concerns
about antibiotic resistance and the harmful effects of residual
antibiotics in foods. However, the removal of current in-feed
antibiotics might increase occurrence of bacterial diseases
including salmonellosis that have been controlled by antibiotics,
as expected in the experiment data or from other countries. Thus,
there is an urgent need to establish a detailed practical guideline
for salmonella management (Jung-Bok Lee. Control of the Recent
Outbreaks of Porcine Salmonella and Proliferative Enteropathy,
Infectious Disease Laboratory, College of Veterinary Medicine,
Konkuk University, Livestock Product Safety. 2010).
These growing concerns have led to a resurgence of interest in
bacteriophage. Seven bacteriophages for control of E. coli O157:H7
are disclosed in U.S. Pat. No. 6,485,902 (2002) and two
bacteriophages for control of various microorganisms are disclosed
in U.S. Pat. No. 6,942,858 (issued to Nymox in 2005). Many
companies have been actively trying to develop various products
using bacteriophages. EBI food system (Europe) developed a food
additive for preventing food poisoning caused by Listeria
monocytogenes, named Listerix-P100, which is the first
bacteriophage product approved by the USFDA. A phage-based product,
LMP-102 was also developed as a food additive against Listeria
monocytogenes, approved as GRAS (Generally Regarded As Safe). In
2007, a phage-based wash produced by OmniLytics was developed to
prevent E. coli 0157 contamination of beef during slaughter,
approved by USDA's Food Safety and Inspection Service (FSIS). In
Europe, Clostridium sporogenes phage NCIMB 30008 and Clostridium
tyrobutiricum phage NCIMB 30008 were registered as feed
preservative against Clostridium contamination of feed in 2003 and
2005, respectively. Such studies show that research into
bacteriophages for the control of antibiotic-unsusceptible bacteria
and contamination of livestock products by zoonotic pathogens is
presently ongoing.
However, most of the phage biocontrol studies have focused on the
control of E. coli, Listeria, and Clostridium. Salmonella is also a
zoonotic pathogen, and damages due to this pathogen are not
reduced. As mentioned above, since Salmonella exhibits multiple
drug resistances, nationwide antimicrobial resistance surveillance
has been conducted in Korea under the Enforcement Decree of the Act
on the Prevention of Contagious Disease (Executive Order 16961),
Enforcement Ordinance of the Act on the Prevention of Contagious
Disease (Ministry of Health and Welfare's Order 179), and
Organization of the National Institute of Health (Executive Order
17164). Accordingly, there is a need for the development of
bacteriophages to control Salmonella.
DISCLOSURE
Technical Problem
In order to overcome problems occurring upon the misuse or overuse
of broad spectrum antibiotics, such as drug resistant bacteria and
drug residues in foods, the present inventors isolated a
bacteriophage from natural sources, in which the bacteriophage has
a specific bactericidal activity against salmonella causing major
diseases in livestock. As a result, they found that the
bacteriophage has a specific bactericidal activity against
Salmonella choleraesuis (SC), Salmonella typhimurium (ST),
Salmonella derby (SD), Salmonella infantis (SI) and Salmonella
newport (SN) with no influences on beneficial bacteria, in addition
to showing excellent acid- and heat-resistance, as identified for
the morphological, biochemical and genetic properties thereof.
Further, they found that the bacteriophage can be applied to
compositions for the prevention or treatment of Salmonella
typhimurium-mediated diseases, such as livestock salmonellosis and
Salmonella food poisoning, and to various products for the
effective prevention and control of Salmonella bacteria
proliferation, including livestock feed additives, drinking water
for livestock, barn sanitizers, and cleaners for meat products,
thereby completing the present invention.
Technical Solution
An object of the present invention is to provide a novel
bacteriophage having a bactericidal activity against Salmonella
choleraesuis.
Another object of the present invention is to provide a composition
for the prevention or treatment of infectious diseases caused by
Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby,
Salmonella infantis or Salmonella newport, comprising the
bacteriophage as an active ingredient.
Still another object of the present invention is to provide an
antibiotic, comprising the bacteriophage as an active
ingredient.
Still another object of the present invention is to provide an
animal feed or drinking water comprising the bacteriophage as an
active ingredient.
Still another object of the present invention is to provide a
sanitizer or cleaner, comprising the bacteriophage as an active
ingredient.
Still another object of the present invention is to provide a
method for preventing or treating of infectious diseases caused by
Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby,
Salmonella infantis or Salmonella newport, using the bacteriophage
or the composition.
Advantageous Effects
The novel bacteriophage of the present invention has a specific
bactericidal activity against Salmonella choleraesuis, Salmonella
typhimurium, Salmonella derby, Salmonella infantis or Salmonella
newport with no influences on beneficial bacteria, and excellent
acid- and heat-resistance and desiccation tolerance. Therefore, the
novel bacteriophage can be used for the prevention or treatment of
salmonellosis or salmonella food poisoning, which is an infectious
disease caused by Salmonella choleraesuis, Salmonella typhimurium,
Salmonella derby, Salmonella infantis or Salmonella newport, and
also widely used in animal feeds, drinking water for livestock,
sanitizers, and cleaners.
DESCRIPTION OF DRAWINGS
FIG. 1 is an electron microscopy photograph of .PHI.CJ11, which
belongs to the family Siphoviridae of morphotype B1, characterized
by an isometric capsid and a long non-contractile tail;
FIG. 2 is a photograph showing the formation of .PHI.CJ11 plaques
in a lawn of salmonella bacteria, in which (A,B; SC, C; SG, D; ST,
E; SI, F,G; SD, H; SN), plaque formation was observed in lawns of
SC, ST, SI, SD and SN, but not in lawns of SG;
FIG. 3 is the result of SDS-PAGE of the isolated bacteriophage
.PHI.CJ11, in which the major proteins were detected at 33 kDa, 55
kDa and 69.5 kDa, and Precision plus protein standard (BIO-RAD) was
used as a marker;
FIG. 4 is the result of PFGE of the isolated bacteriophage
.PHI.CJ11, in which a total genome size of .PHI.CJ11 was
approximately 140 kbp, and the CHEF DNA Size Standard Lambda Ladder
(Bio-Rad) was used as a DNA size marker;
FIG. 5 is the result of PCR, performed using each primer set for
the .PHI.CJ11 genomic DNA, in which A; a primer set of SEQ ID NOs.
5 and 6, B; a primer set of SEQ ID NOs. 7 and 8, C; a primer set of
SEQ ID NOs. 9 and 10, and D; a primer set of SEQ ID NOs. 11 and 12,
and all of A, B, C and D lanes had PCR products of approximately 1
kbp or more to 2 kbp or less;
FIG. 6 is the result of acid-resistance assay on the bacteriophage
.PHI.CJ11, showing the number of surviving bacteriophage at pH 2.1,
2.5, 3.0, 3.5, 4.0, 5.5, 6.4, 6.9, 7.4, 8.0, 9.0, 9.8 and 11.0, in
which the bacteriophage .PHI.CJ11 did not lose its activity until
pH 5.5, but the bacteriophage .PHI.CJ11 showed reduced activity at
pH 4 and pH 3.5, and completely lost its activity at pH 3.0 or
lower, as compared to a control;
FIG. 7 is the result of heat-resistance assay on the bacteriophage
.PHI.CJ11, showing the number of surviving bacteriophage at 37, 45,
53, 60, and 70.degree. C. for 0, 10, 30, 60 and 120 minutes, in
which the bacteriophage .PHI.CJ11 maintained its activity even
though exposed to 60.degree. C. for up to 2 hours;
FIG. 8 is the result of desiccation tolerance assay on the
bacteriophage .PHI.CJ11 dried with the aid of a SpeedVac
concentrator, in which when titer changes under the dry condition
were measured in comparison with pre-drying titers, the activity
was maintained at 60.degree. C. for up to 1 hour; and
FIG. 9 is the results of body weight changes due to toxicity after
single oral administration of Sprague-Dawley rats with .PHI.CJ11,
in which observation of body weight changes before and 1, 3, 7, 10
and 14 days after administration with .PHI.CJ11 showed no
significant changes in comparison with the control group
(.box-solid.; male control, .quadrature.; .PHI.CJ11 male,
.circle-solid.; female control, .smallcircle.; .PHI.CJ11
female).
BEST MODE
In one aspect to achieve the above objects, the present invention
provides a novel bacteriophage having a specific bactericidal
activity against Salmonella choleraesuis, Salmonella typhimurium,
Salmonella derby, Salmonella infantis or Salmonella newport.
The present inventors collected fecal and sewage samples from
swinery, and isolated therefrom bacteriophages that can lyse the
host cell SC. They were also found that these bacteriophages can
lyse ST, SD, SI and SN (FIG. 2 and Table 1). A morphological
examination under an electron microscope confirmed that the
bacteriophage (.PHI.CJ11) belongs to the family Siphoviridae of
morphotype B1 (FIG. 1). Further, the bacteriophage .PHI.CJ11 was
found to have major structural proteins of approximately 69.5 kDa,
55 kDa and 33 kDa, as measured by a protein pattern analysis (FIG.
3), and a genome analysis showed that .PHI.CJ11 has a total genome
size of approximately 97-145.5 kbp (FIG. 4). Furthermore, the
results of analyzing its genetic features showed that the
bacteriophage includes nucleic acid molecules represented by SEQ ID
NOs. 1 to 4 within the total genome (Example 6). Based on SEQ ID
NOs. 1 to 4, genetic similarity with other species was compared. It
was found that the bacteriophage showed very low genetic similarity
with the known bacteriophages, indicating that the bacteriophage is
a novel bacteriophage (Table 2). For more detail analysis of
genetic features, the .PHI.CJ11-specific primer sets, namely, SEQ
ID NOs. 5 and 6, SEQ ID NOs. 7 and 8, SEQ ID NOs. 9 and 10, and SEQ
ID NOs. 11 and 12 were used to perform PCR. Each PCR product was
found to have a size of 1.4 kbp, 1.2 kbp, 1.25 kbp and 1.5 kbp
(FIG. 5).
Meanwhile, when SC, ST, SD, SI and SN were infected with .PHI.CJ11,
the phage plaques (clear zone on soft agar created by host cell
lysis of one bacteriophage) were observed (FIG. 2). The stability
of .PHI.CJ11 was examined under various temperature and pH
conditions, resulting in that .PHI.CJ11 stably maintains in a wide
range of pH environments from pH 3.5 to pH 11.0 (FIG. 6) and in
high temperature environments from 37.degree. C. to 70.degree. C.
(FIG. 7), and even after desiccation (FIG. 8). Also, the wild-type
strains SC, ST, SD, SI and SN were also found to fall within the
host cell range of .PHI.CJ11 (Table 3).
Finally, the results of dermal and ocular irritation tests on
.PHI.CJ11 in specific-pathogen-free (SPF) New Zealand White rabbits
showed that the primary irritation index (PII) was 0.33, indicating
no irritant, and the index of acute ocular irritation (IAOI) was 0
in washing and non-washing groups during the whole experimental
periods, indicating no irritant. The results of oral administration
of .PHI.CJ11 showed no changes in weight gain (FIG. 9). As well,
mortality, general symptoms (Table 4) and organ abnormality (Table
5) were not observed, indicating no toxicity.
Accordingly, the present inventors designated the bacteriophage as
"Bacteriophage .PHI.CJ11", in which the bacteriophage was isolated
from fecal and sewage samples from swinery and has a specific
bactericidal activity against SC, ST, SD, SI and SN and the above
characteristics, and deposited at the Korean Culture Center of
Microorganisms (361-221, Honje 1, Seodaemun, Seoul) on Sep. 9, 2011
under accession number KCCM11208P.
In another aspect to achieve the above objects, the present
invention provides a composition for the prevention or treatment of
infectious disease caused by one or more Salmonella bacteria
selected from the group consisting of Salmonella choleraesuis,
Salmonella typhimurium, Salmonella derby, Salmonella infantis and
Salmonella newport, comprising the bacteriophage as an active
ingredient.
Having specific bactericidal activity against Salmonella
choleraesuis, Salmonella typhimurium, Salmonella derby, Salmonella
infantis and Salmonella newport, the bacteriophage of the present
invention may be used for the purpose of preventing or treating the
diseases caused by these bacteria. Preferably, examples of the
infectious diseases include porcine salmonellosis and Salmonella
food poisoning caused by Salmonella choleraesuis or Salmonella
typhimurium, and acute or chronic porcine enteritis caused by
Salmonella derby, Salmonella infantis, Salmonella newport, but are
not limited thereto.
As used herein, the term "salmonellosis" refers to symptoms
following salmonella infection, such as fever, headache, diarrhea,
and vomiting. That is, salmonellosis is an infection with bacteria
of the genus Salmonella, which is defined with two clinical forms:
an acute septicemic form that resembles typhoid fever and an acute
gastroenteritis, including enteritis, food poisoning, and acute
septicemia.
As used herein, the term "prevention" means all of the actions in
which disease progress is restrained or retarded by the
administration of the composition.
As used herein, the term "treatment" means all of the actions in
which the condition has taken a turn for the better or been
restrained or modified favorably by the administration of the
composition.
The composition of the present invention includes .PHI.CJ11 in an
amount of 1.times.10.sup.2 to 1.times.10.sup.12 PFU/mL, and
preferably in an amount of 1.times.10.sup.6 to 1.times.10.sup.10
PFU/mL.
On the other hand, the composition of the present invention may
further include a pharmaceutically acceptable carrier.
As used herein, the term "pharmaceutically acceptable carrier"
refers to a carrier or diluent that does not cause significant
irritation to an organism and does not abrogate the biological
activity and properties of the administered compound. For
formulation of the composition into a liquid preparation, a
pharmaceutically acceptable carrier which is sterile and
biocompatible may be used such as saline, sterile water, Ringer's
solution, buffered physiological saline, albumin infusion solution,
dextrose solution, maltodextrin solution, glycerol, ethanol, and
mixtures of one or more thereof. If necessary, other conventional
additives such as antioxidants, buffers, and bacteriostatic agents
may be added. Further, diluents, dispersants, surfactants, binders
and lubricants may be additionally added to the composition to
prepare injectable formulations such as aqueous solutions,
suspensions, and emulsions, or oral formulations such as pills,
capsules, granules, or tablets.
The prophylactic or therapeutic compositions of the present
invention may be applied or sprayed to the afflicted area, or
administered by oral or parenteral routes. The parenteral
administration may include intravenous, intraperitoneal,
intramuscular, subcutaneous or topical administration.
The dosage suitable for applying, spraying, or administrating the
composition of the present invention will depend upon a variety of
factors including formulation method, the mode of administration,
the age, weight, sex, condition, and diet of the patient or animal
being treated, the time of administration, the route of
administration, the rate of excretion, and reaction sensitivity. A
physician or veterinarian having ordinary skill in the art can
readily determine and prescribe the effective amount of the
composition required.
Examples of the oral dosage forms including the composition of the
present invention as an active ingredient include tablets, troches,
lozenges, aqueous or emulsive suspensions, powder or granules,
emulsions, hard or soft capsules, syrups, or elixirs. For
formulation such as tablets and capsules, the following are useful:
a binder such as lactose, saccharose, sorbitol, mannitol, starch,
amylopectin, cellulose or gelatin; an excipient such as dicalcium
phosphate; a disintegrant such as corn starch or sweet potato
starch; and a lubricant such as magnesium stearate, calcium
stearate, sodium stearylfumarate, or polyethylene glycol wax. For
capsules, a liquid carrier such as lipid may be further used in
addition to the above-mentioned compounds.
The parenteral dosage forms including the composition of the
present invention as an active ingredient may be formulated into
injections via subcutaneous, intravenous, or intramuscular routes,
suppositories, or sprays inhalable via the respiratory tract, such
as aerosols. Injection forms may be prepared by dissolving or
suspending the composition of the present invention, together with
a stabilizer or a buffer, in water and loading the solution or
suspension onto ampules or vial unit forms. For sprays, such as
aerosols, a propellant for spraying a water-dispersed concentrate
or wetting powder may be used in combination with an additive.
In still another aspect to achieve the above objects, the present
invention provides an antibiotic comprising the bacteriophage as an
active ingredient.
As used herein, the term "antibiotic" means any drug that is
applied to animals to kill pathogens, and used herein as a general
term for antiseptics, bactericidal agents and antibacterial agents.
The animals are mammals including human. The bacteriophage of the
present invention, unlike the conventional antibiotics, has a high
specificity to Salmonella so as to kill the specific pathogens
without affecting beneficial bacteria, and does not induce drug
resistance so that it can be provided as a novel antibiotic with a
comparatively long life cycle.
In still another aspect to achieve the above objects, the present
invention provides an animal feed or drinking water comprising the
bacteriophage as an active ingredient.
In-feed antibiotics used in the livestock and fishery industries
are intended to prevent infections. However, most of the currently
available in-feed antibiotics are problematic in that they are apt
to induce the occurrence of resistant strains and may be
transferred to humans, due to remaining in livestock products. The
uptake of such residual antibiotics may make human pathogens
resistant to antibiotics, resulting in the spread of diseases. In
addition, since there are a variety of in-feed antibiotics, the
increasing global emergence of multidrug-resistant strain is a
serious concern. Therefore, the bacteriophage of the present
invention can be used as an in-feed antibiotic that is more
eco-friendly and able to solve the above problems.
The animal feed of the present invention may be prepared by adding
the bacteriophage directly or in separate feed additive form to an
animal feed. The bacteriophage of the present invention may be
contained in the animal feed as a liquid or in a solid form,
preferably in a dried powder. The drying process may be performed
by air drying, natural drying, spray drying, and freeze-drying, but
is not limited thereto. The bacteriophage of the present invention
may be added as a powder form in an amount of 0.05 to 10% by
weight, preferably 0.1 to 2% by weight, based on the weight of
animal feed. The animal feed may also include other conventional
additives for long-term preservation, in addition to the
bacteriophage of the present invention.
The feed additive of the present invention may additionally include
other non-pathogenic microorganisms. The available additional
microorganism may be selected from the group consisting of Bacillus
subtilis that can produce protease, lipase and invertase,
Lactobacillus sp. strain that can exert physiological activity and
a function of decomposing under anaerobic conditions, such as in
the stomach of cattle, filamentous fungi including Aspergillus
oryzae (J Animal Sci 43:910-926, 1976) that increases the weight of
domestic animals, enhances milk production and helps the digestion
and absorptiveness of feeds, and yeast including Saccharomyce
scerevisiae (J Anim Sci 56:735-739, 1983).
The feed including .PHI.CJ11 of the present invention may include
plant-based feeds, such as grain, nut, food byproduct, seaweed,
fiber, drug byproduct, oil, starch, meal, and grain byproduct, and
animal-based feeds such as protein, inorganic matter, fat, mineral,
fat, single cell protein, zooplankton, and food waste, but is not
limited thereto.
The feed additive including .PHI.CJ11 of the present invention may
include binders, emulsifiers, and preservatives for the prevention
of quality deterioration, amino acids, vitamins, enzymes,
probiotics, flavorings, non-protein nitrogen, silicates, buffering
agents, coloring agents, extracts, and oligosaccharides for
efficiency improvement, and other feed premixtures, but is not
limited thereto.
Further, the supply of drinking water mixed with the bacteriophage
of the present invention can reduce the number of Salmonella
bacteria in the intestine of livestock, thereby obtaining
Salmonella-free livestock.
In still another aspect to achieve the above objects, the present
invention provides a sanitizer or cleaner comprising the
bacteriophage as an active ingredient.
In still another aspect to achieve the above objects, the present
invention provides a method for treating infectious diseases caused
by Salmonella choleraesuis, Salmonella typhimurium, Salmonella
derby, Salmonella infantis or Salmonella newport using the
bacteriophage or the composition.
In detail, the therapeutic method of the present invention
comprises the step of administering a pharmaceutically effective
amount of the bacteriophage or the composition to an individual
having infectious diseases caused by Salmonella choleraesuis,
Salmonella typhimurium, Salmonella derby, Salmonella infantis or
Salmonella newport.
The bacteriophage or the composition of the present invention may
be administered in the form of a pharmaceutical formulation into
animals or may be ingested as a mixture with animal feed or
drinking water by animals and preferably as a mixture with animal
feed.
As long as it reaches target tissues, any route, whether oral or
parenteral, may be taken for administering the bacteriophage or the
composition of the present invention. In detail, the composition of
the present invention may be administered in a typical manner via
any route such as oral, rectal, topical, intravenous,
intraperitoneal, intramuscular, intraarterial, transdermal,
intranasal, and inhalation routes.
It will be obvious to those skilled in the art that the total daily
dose of the bacteriophage or the composition of the present
invention to be administered by the therapeutic method should be
determined through appropriate medical judgment by a physician.
Preferably, the therapeutically effective amount for given patients
may vary depending on various factors well known in the medical
art, including the kind and degree of the response to be achieved,
the patient's condition such as age, body weight, state of health,
sex, and diet, time and route of administration, the secretion rate
of the composition, the time period of therapy, concrete
compositions according to whether other agents are used therewith
or not, etc.
MODE FOR INVENTION
Hereinafter, the present invention will be described in more detail
with reference to Examples. However, these Examples are for
illustrative purposes only, and the invention is not intended to be
limited by these Examples.
Example 1: Salmonella Bacteriophage Isolation
Example 1-1: Bacteriophage Screening and Single
Bacteriophage Isolation
50 mL of sample from swinery and sewage effluent was transferred to
a centrifuge tube, and centrifuged at 4000 rpm for 10 minutes.
Then, the supernatant was filtered using a 0.45 .mu.m filter. 18 mL
of sample filtrate was mixed with 150 .mu.l of Salmonella
choleraesuis ("SC") shaking culture medium (OD.sub.600=2) and 2 mL
of 10.times. Luria-Bertani medium (tryptone 10 g/L, yeast extract 5
g/L and NaCl 10 g/L: LB medium). The mixture was cultured at
37.degree. C. for 18 hours, and the culture medium was centrifuged
at 4000 rpm for 10 minutes. The supernatant was filtered using a
0.2 .mu.m filter. 3 mL of 0.7% agar (w/v) and 150 .mu.l of SC
shaking culture medium (OD.sub.600=2) were mixed, and plated onto
LB plate ("top-agar"), and allowed to solidify. 10 .mu.l of the
culture filtrate was spread thereon, and cultured for 18 hours at
37.degree. C., and the titration of phage lysate was performed on
the top-agar, called soft agar overlay method.
The sample culture medium containing the phage lysate was properly
diluted, and mixed with 150 .mu.l of SC shaking culture medium
(OD.sub.600=2), followed by soft agar overlay method, so that
single plaques were obtained. A single plaque represents one
bacteriophage and thus, for isolation of single bacteriophages, one
phage plaque was added to 400 .mu.l of SM solution (NaCl, 5.8 g/L,
MgSO.sub.47H.sub.2O, 2 g/L, 1 M Tris-Cl (pH 7.5) 50 ml/L), and left
for 4 hours at room temperature to isolate single bacteriophages.
To purify the bacteriophage in large quantities, 100 .mu.l of
supernatant was taken from the single bacteriophage solution, and
mixed with 12 mL of 0.7% agar and 500 .mu.l of SC shaking culture
medium, followed by soft agar overlay method on LB plate having a
diameter of 150 mm. When lysis was completed, 15 mL of SM solution
was added to the plate. The plate was gently shaken for 4 hours at
room temperature to elute the bacteriophages from the top-agar. The
SM solution containing the eluted bacteriophages was recovered,
chloroform was added to a final volume of 1%, and mixed well for 10
minutes. The solution was centrifuged at 4000 rpm for 10 minutes.
The obtained supernatant was filtered using a 0.45 .mu.m filter,
and stored in the refrigerator.
Example 1-2: Large-Scale Batches of Bacteriophage
The selected bacteriophages were cultured in large quantities using
SC. SC was shaking-cultured, and an aliquot of 1.5.times.10.sup.10
cfu (colony forming units) was centrifuged at 4000 rpm for 10
minutes, and the pellet was resuspended in 4 ml of SM solution. The
bacteriophage of 9.0.times.10.sup.8 PFU (plaque forming unit) was
inoculated thereto (MOI: multiplicity of infection=0.001), and left
at 37.degree. C. for 20 minutes. The solution was inoculated into
150 ml of LB media, and cultured at 37.degree. C. for 5 hours.
Chloroform was added to a final volume of 1%, and the culture
solution was shaken for 20 minutes. DNase I and RNase A were added
to a final concentration of 1 .mu.g/ml, respectively. The solution
was left at 37.degree. C. for 30 minutes. NaCl and PEG
(polyethylene glycol) were added to a final concentration of 1 M
and 10% (w/v), respectively and left at 4.degree. C. for an
additional 3 hours. The solution was centrifuged at 4.degree. C.
and 12,000 rpm for 20 minutes to discard the supernatant. The
pellet was resuspended in 5 mL of SM solution, and left at room
temperature for 20 minutes. 4 mL of chloroform was added thereto
and mixed well, followed by centrifugation at 4.degree. C. and 4000
rpm for 20 minutes. The supernatant was filtered using a 0.2 .mu.m
filter, and the bacteriophage was purified by glycerol density
gradient ultracentrifugation (density: 40%, 5% glycerol at 35,000
rpm and 4.degree. C. for 1 hour). The purified bacteriophage was
designated as "Bacteriophage .PHI.CJ11", and resuspended in 300
.mu.l of SM solution, followed by titration. The bacteriophage
.PHI.CJ11 was deposited at the Korean Culture Center of
Microorganisms (361-221, Honje 1, Seodaemun, Seoul) on Sep. 9, 2011
under accession number KCCM11208P.
Example 2: Examination on .PHI.CJ11 Infection of Salmonella
To analyze the selected bacteriophage for lytic activity on
Salmonella species other than SC, attempts were made of cross
infection with other Salmonella species. As a result, .PHI.CJ11
infected SC (Salmonella choleraesuis), ST (Salmonella typhimurium),
SD (Salmonella derby), SN (Salmonella newport), SI (Salmonella
infantis), SA (Salmonella arizonae) and SB (Salmonella bongori),
but did not infect SE (Salmonella enteritidis), SG (Salmonella
gallinarum), and SP (Salmonella pullorum) (Table 1 and FIG. 2).
TABLE-US-00001 TABLE 1 .PHI.CJ11 Infection of Salmonella Phage
plaque Serotype Strain name formation SC ATCC 10708 .largecircle.
SN SL 317 .largecircle. SD ATCC 2468 .largecircle. SE SGSC 2282 X
SG SGSC 2293 X SA ATCC 12398 .largecircle. SB ATCC 12397
.largecircle. ST 13 .largecircle. SI SARB 26 .largecircle. SP SGSC
2295 X * ATCC: American Type Culture Collection * SGSC: Salmonella
Genetic Stock Center
Moreover, FIG. 2 is a photograph showing the formation of .PHI.CJ11
plaques in a lawn of salmonella bacteria. As shown in FIG. 2 (A,B;
SC, C; SG, D; ST, E; SI, F,G; SD, H; SN), plaque formation was
observed in lawns of SC, ST, SI, SD and SN, but not in lawns of
SG.
Example 3: Morphology of .PHI.CJ11
The purified .PHI.CJ11 was diluted in the SM buffer solution, and
then mounted on a copper grid, stained with 2% uranyl acetate for 3
to 5 seconds, and dried. Examination under a transmission electron
microscope (LIBRA 120, Carl Zeiss transmission electron Microscope,
80 kV, magnification of .times.120,000.times.200,000) was performed
(FIG. 1). FIG. 1 is an electron microscopy photograph of .PHI.CJ11.
As shown in FIG. 1, it was found that the purified .PHI.CJ11
belongs to the family Siphoviridae of morphotype B1, characterized
by an isometric capsid and a long non-contractile tail.
Example 4: Protein Pattern Analysis of .PHI.CJ11
15 .mu.L of a .PHI.CJ11 solution purified at a titer of 10.sup.11
PFU/mL was mixed with 3 .mu.L of a 5.times.SDS sample solution, and
heated for 5 minutes. 12% SDS-PAGE was performed, and then the gel
was stained with Coomassie blue for 1 hour at room temperature
(FIG. 3). FIG. 3 is the result of SDS-PAGE of the isolated
bacteriophage .PHI.CJ11, in which Precision plus protein standard
(BIO-RAD) was used as a marker. As shown in FIG. 3, the major
proteins were detected at 33 kDa, 55 kDa and 69.5 kDa.
Example 5: Analysis of Total Genomic DNA Size of .PHI.CJ11
Genomic DNA of the purified .PHI.CJ11 was isolated using
ultracentrifugation. In detail, to the purified .PHI.CJ11 culture
medium were added EDTA (ethylenediaminetetraacetic acid (pH 8.0)),
proteinase K, and SDS (sodium dodecyl sulfate) at a final
concentration of 20 mM, 50 ug/mL, and 0.5% (w/v), respectively,
followed by incubation at 50.degree. C. for 1 hour. An equal volume
of phenol (pH 8.0) was added and mixed well. After centrifugation
at room temperature and 12,000 rpm for 10 minutes, the supernatant
was mixed well with an equal volume of PC (phenol:chloroform=1:1).
Another centrifugation was performed at room temperature and 12,000
rpm for 10 minutes. Then, a supernatant was obtained, and mixed
with an equal volume of chloroform, followed by centrifugation at
room temperature and 12,000 rpm for 10 minutes. The obtained
supernatant was mixed with 1/10 volume of 3 M sodium acetate and
two volumes of cold 95% ethanol, and left at -20.degree. C. for 1
hour. After centrifugation at 0.degree. C. and 12,000 rpm for 10
minutes, the supernatant was completely removed, and the DNA pellet
was dissolved in 50 .mu.L of TE (Tris-EDTA (pH 8.0)). The extracted
DNA was diluted 10-fold, and measured for absorbance at OD.sub.260
to determine its concentration. 1 .mu.g of the total genomic DNA
was loaded onto 1' PFGE (pulse-field gel electrophoresis) agarose
gel, and electrophoresed at 14.degree. C. for 22 hours in a BIORAD
CHEF DR II PFGE system under the conditions of switch time ramp for
50-90 seconds, 6 V/cm (200V). The CHEF DNA Size Standard Lambda
Ladder (Bio-Rad) was used as a DNA size marker (FIG. 4). FIG. 4 is
the result of PFGE of the isolated bacteriophage .PHI.CJ11. As
shown in FIG. 4, DNA of approximately 140 kbp present between 48.5
to 1,000 kbp was observed.
Example 6: Genetic Analysis of .PHI.CJ11
For the genetic analysis of the purified .PHI.CJ11, 5 .mu.g of the
genomic DNA of .PHI.CJ11 was double digested with the restriction
enzymes PstI, XbaI and BamHI, EcoRI and SalI. The vector pCL1920
(Promega) was digested with the restriction enzymes PstI, XbaI and
BamHI, EcoRI and SalI, and then treated with CIP (calf intestinal
alkaline phosphatase). The digested genomic DNA was mixed at a
ratio of 3:1 with the vector, and ligated at 16.degree. C. for 2
hours. The resulting recombinant vector was transformed into E.
coli DH5a which was then plated on an LB plate containing
specinomycin and X-gal
(5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) for selection
of blue/white colonies. The selected colonies were cultured for 16
hours in a culture medium containing the antibiotic with shaking.
Then, plasmids were extracted using a Plasmid purification kit
(Promega).
The cloning of the plasmids was confirmed by PCR using primer sets
of FTR135 and FTR136 (SEQ ID NOs. 13 and 14) and selection was made
only of insert fragments having a size of 1 kb or longer. Their
nucleotide sequences were analyzed using the primer sets. The
nucleotide sequences thus obtained were given in SEQ ID NOs. 1 to
4, respectively, each having a size of 1 to 2 kbp or less, and
analyzed for sequence similarity with the aid of NCBI blastx and
blastn program, and the results are summarized in Table 2,
below.
TABLE-US-00002 TABLE 2 Comparison of Sequence Similarity between
.PHI.CJ11 and Other Bacteriophages Blastx Organism Protein Query
Subject Identity e-value 1 Enterobacteria hypothetical protein
436-627 1-64 63/64 7e-32 phage T5 (98%) Enterobacteria hypothetical
protein 10-423 1-139 127/139 3e-31 phage SPC35 (91%) Enterobacteria
hypothetical protein 3-863 30-315 264/291 2e-116 phage SPC35 (91%)
Enterobacteria hypothetical protein 3-863 30-315 255/291 1e-113
phage T5 (88%) Enterobacteria hypothetical protein 3-761 29-281
235/253 7e-107 phage EPS7 (93%) Klebsiella phage hypothetical
protein 9-761 22-276 217/255 4e-100 KP15 (85%) Enterobacteria
putative SPFH domain- 9-839 18-257 223/281 7e-99 phage RB43
containing protein (79%) Enterobacteria hypothetical protein 9-761
18-272 219/255 2e-98 phage RB16 (86%) 2 Enterobacteria hypothetical
protein 412-771 1-120 109/120 7e-57 phage EPS7 (91%) Enterobacteria
hypothetical protein 490-771 1-94 94/94 3e-46 phage T5 (100%)
Enterobacteria D11 protein 532-2 1-177 175/177 5e-96 phage T5 (99%)
Enterobacteria D11 protein 532-2 1-177 174/177 2e-95 phage SPC35
(98%) Enterobacteria D11 protein 532-8 1-175 163/175 4e-90 phage
EPS7 (93%) Enterobacteria hypothetical protein 872-528 5-120 96/116
2e-46 phage EPS7 (83%) 3 Enterobacteria tail protein Pb4 777-4
1-258 221/258 4e-128 phage SPC35 (86%) Enterobacteria tail protein
Pb4 777-4 1-258 197/258 6e-115 phage T5 (76%) Enterobacteria tail
protein Pb3 1322-780 769-949 178/181 2e-96 phage SPC35 (98%)
Enterobacteria tail protein Pb3 1322-780 769-949 160/181 9e-88
phage EPS7 (88%) Enterobacteria structural tail 1322-780 769-949
156/181 8e-84 phage T5 protein Pb3 (86%) Enterobacteria tail
protein Pb4 423-4 1-140 112/140 2e-62 phage EPS7 (80%) 4
Enterobacteria flap endonuclease 980-564 153-291 138/139 1e-73
phage SPC35 (99%) Enterobacteria flap endonuclease 980-564 153-291
138/139 1e-73 phage T5 (99%) Enterobacteria flap endonuclease
980-564 153-291 135/139 4e-72 phage EPS7 (97%) Enterobacteria
putative deoxyUTP 564-157 1-136 130/136 3e-70 phage SPC35
pyrophosphatase (96%) Enterobacteria putative deoxyUTP 564-160
1-135 130/135 2e-69 phage T5 pyrophosphatase (96%) Enterobacteria
putative deoxyUTP 564-157 1-136 128/136 2e-67 phage EPS7
pyrophosphatase (94%)
Example 7: Design of .PHI.CJ11-Specific Primer Sequences
In order to identify .PHI.CJ11, .PHI.CJ11-specific primers were
designed on the basis of SEQ ID NOS. 1 to 4. PCR was performed
using each primer set of SEQ ID NOS. 5 and 6, SEQ ID NOs. 7 and 8,
SEQ ID NOs. 9 and 10, and SEQ ID NOs. 11 and 12. 0.1 .mu.g of the
genomic DNA of bacteriophage and 0.5 pmol of each primer were added
to a pre-mix (Bioneer), and the final volume was adjusted to 20
.mu.L. PCR was performed with 30 cycles of denaturation; 94.degree.
C. 30 seconds, annealing; 55.degree. C. 30 seconds, and
polymerization; 72.degree. C., 1.5 minutes (FIG. 5). FIG. 5 is the
result of PCR, performed using each primer set for the .PHI.CJ11
genomic DNA. A; a primer set of SEQ ID NOs. 5 and 6, B; a primer
set of SEQ ID NOs. 7 and 8, C; a primer set of SEQ ID NOs. 9 and
10, and D; a primer set of SEQ ID NOs. 11 and 12. All of A, B, C
and D lanes had PCR products of approximately 1 to 2 kbp. As shown
in FIG. 5, the PCR products thus obtained had a size of
approximately 1 kbp or more to 2 kbp or less, with the primer sets
of SEQ ID NOs. 5 and 6, SEQ ID NOs. 7 and 8, SEQ ID NOs. 9 and 10,
and SEQ ID NOs. 11 and 12.
Example 8: pH Stability of Bacteriophage
In order to determine whether .PHI.CJ11 survives with stability
under the low pH environment in the stomach of pig, .PHI.CJ11 was
assayed for stability in a wide range of pH (pH 2.1, 2.5, 3.0, 3.5,
4.0, 5.5, 6.4, 6.9, 7.4, 8.2, 9.0, 9.8 and 11.0). Various pH
solutions (sodium acetate buffer (pH 2.1, pH 4.0, pH 5.5, and pH
6.4), sodium citrate buffer (pH 2.5, pH 3.0, and pH 3.5), sodium
phosphate buffer (pH 6.9 and pH 7.4) and Tris-HCl (pH 8.2, pH 9.0,
pH 9.8 and pH 11.0)) were prepared to have a concentration of 0.2
M. 180 .mu.L of each pH solution was mixed with 20 .mu.L of a
bacteriophage solution (1.0.times.10.sup.11 PFU/mL) to give each pH
solution a concentration of 1 M, followed by incubation at room
temperature for 2 hours. The reaction solution was serially
diluted, and 10 .mu.L of each dilution was cultured at 37.degree.
C. for 18 hours by a soft agar overlay method to determine the
titers of the phage lysates (FIG. 6). FIG. 6 is the result of
acid-resistance assay on the bacteriophage .PHI.CJ11, showing the
number of surviving bacteriophage at pH 2.1, 2.5, 3.0, 3.5, 4.0,
5.5, 6.4, 6.9, 7.4, 8.0, 9.0, 9.8 and 11.0. The bacteriophage
.PHI.CJ11 did not lose its activity until pH 5.5. However, the
bacteriophage .PHI.CJ11 showed reduced activity at pH 4 and pH 3.5,
and completely lost its activity at pH 3.0 or lower, as compared to
a control. As shown in FIG. 6, the bacteriophage did not lose its
activity and remained stable down to pH 5.5 whereas it lost its
activity at pH 3.0 or lower.
Example 9: Heat Stability of Bacteriophage
For use as a feed additive, the bacteriophage was assayed for
stability to the heat generated during a formulation process. In
this regard, 200 .mu.L of a .PHI.CJ11 solution with a titer of
1.0.times.10.sup.11 PFU/mL was incubated at 37.degree. C.,
45.degree. C., 53.degree. C., 60.degree. C., and 70.degree. C. for
0 minute, 10 minutes, 30 minutes, 60 minutes and 120 minutes. The
solution was serially diluted, and 10 .mu.L of each dilution was
cultured at 37.degree. C. for 18 hours by a soft agar overlay
method to determine the titers of phage lysates (FIG. 7). FIG. 7 is
the result of heat-resistance assay on the bacteriophage .PHI.CJ11,
showing the number of surviving bacteriophage at 37.degree. C.,
45.degree. C., 53.degree. C., 60.degree. C., and 70.degree. C. for
0, 10, 30, 60 and 120 minutes. As shown in FIG. 7, the
bacteriophage .PHI.CJ11 maintained its activity even though exposed
at 60.degree. C. up to 2 hours.
Example 10: Desiccation Tolerance of Bacteriophage
For use as a feed additive, the bacteriophage .PHI.CJ11 was assayed
for tolerance to the dry condition set for a formulation process.
On the basis of the results obtained from the heat stability assay,
a desiccation assay was performed using a SpeedVac concentrator.
200 .mu.L of a .PHI.CJ11 solution having a titer of
1.0.times.10.sup.11 PFU/mL was dried under vacuum at 60.degree. C.
for 2 hours, and the pellet thus obtained was completely
re-suspended in 200 .mu.L of the SM solution at 4.degree. C. for
one day, and measured for titer values (FIG. 8). FIG. 8 is the
result of desiccation tolerance assay on the bacteriophage
.PHI.CJ11 dried with the aid of a SpeedVac concentrator. As shown
in FIG. 8, when titer changes under the dry condition were measured
in comparison with pre-drying titers, the activity was maintained
at 60.degree. C. up to 1 hour.
Example 11: Infection Spectrum of Bacteriophage
.PHI.CJ11 was assayed for lytic activity against the wild-type (2
strains), Salmonella choleraesuis (5 strains), Salmonella
typhimurium (17 strains), Salmonella infantis (4 strains),
Salmonella newport (6 strains), Salmonella derby (2 strains) and
Salmonella dublin (3 strains), obtained from Laboratory of Avian
Diseases, College of Veterinary Medicine, Seoul National
University, in addition to SC (ATCC SC10708) used in the
experiment. 150 .mu.L of each strain shaking culture medium
(OD.sub.600=2) was mixed, and 10 .mu.L of .PHI.CJ11 solution having
a titer of 10'PFU/mL was cultured at 37.degree. C. for 18 hours
using a soft agar overlay method to monitor the formation of
plaques (Table 3). Formation of phage plaque was observed in 8
strains of SC.
TABLE-US-00003 TABLE 3 Wild-type strains SC, ST, SD, SI, SN
infected by .PHI.CJ11 Phage plaque Phage plaque Serotype Strain
name formation serotype Strain name formation SC S. choleraesuis
ATCC .smallcircle. SI S. infantis SARB 26 .smallcircle. 2929 S.
choleraesuis ATCC .smallcircle. S. infantis SARB 27 .smallcircle.
2930 S. choleraesuis ATCC .smallcircle. S. infantis S1326/28
.smallcircle. 2932 S. choleraesuis ATCC .smallcircle. S. infantis
B09-106 .smallcircle. 2933 S. choleraesuis ATCC .smallcircle. SN S.
newport SARB 36 .smallcircle. 2425 S. choleraesuis ATCC
.smallcircle. S. newport SARB 37 .smallcircle. 10708 S.
choleraesuis SNU#1 .smallcircle. S. newport SARB 38 .smallcircle.
S. choleraesuis SNU#2 .smallcircle. S. newport 7257 .smallcircle.
ST S. typhimurium SNU .smallcircle. S. newport SL 317 .smallcircle.
ST1 S. typhimurium SNU .smallcircle. S. newport SL 254
.smallcircle. ST2 S. typhimurium SNU .smallcircle. SD S. derby ATCC
.smallcircle. ST4 2466 S. typhimurium SNU .smallcircle. S. derby
ATCC .smallcircle. ST7 2468 S. typhimurium SNU .smallcircle. SD S.
dublin SA 4405 .smallcircle. ST8 S. typhimurium SNU .smallcircle.
S. dublin RKS 4699 .smallcircle. ST11 S. typhimurium SNU
.smallcircle. S. dublin 88/6 .smallcircle. ST12 S. typhimurium SNU
.smallcircle. SA S. arizonae ATCC .smallcircle. ST13 12398 S.
typhimurium SNU .smallcircle. SB S. bongori ATCC .smallcircle. ST14
12397 S. typhimurium SNU .smallcircle. SH S. heidelberg SARA 33
.smallcircle. ST17 S. typhimurium SNU .smallcircle. S. heidelberg
SARA 23 .smallcircle. ST18 S. typhimurium SNU .smallcircle. SM S.
maimi SARB 28 .smallcircle. ST19 S. typhimurium SNU .smallcircle.
S. maimi SARB 29 .smallcircle. ST20 S. typhimurium SNU
.smallcircle. SP S. panama SARB 39 .smallcircle. ST26 S.
typhimurium SNU .smallcircle. S. panama SARB 40 .smallcircle. ST38
S. typhimurium SNU .smallcircle. S. panama SARB 41 .smallcircle.
ST41 S. typhimurium SNU .smallcircle. S. panama 7261 .smallcircle.
ST42 SGSC: Salmonella genetic stock center ATCC: American Type
Culture Collection SNU: Laboratory of Avian Diseases, College of
Veterinary Medicine, Seoul National University
Example 12: Toxicity Assay of Bacteriophage
Dermal and ocular irritation tests were performed in
specific-pathogen-free (SPF) New Zealand white rabbits, which are
commonly used in the toxicity test of bacteriophage .PHI.CJ11 for
the prevention of salmonellosis and salmonella food poisoning, and
of which experimental data were accumulated to allow easy analysis
of experiment results. The normal abdominal skin (non-injured skin)
and injured abdominal skin of rabbits were covered and contacted
with 2.5 cm.times.2.5 cm of gauze applied with the test substance,
and each 0.5 mL/site was applied. No changes in general symptoms
were observed, and a slight weight loss was observed 1 day after
application of the test substance, which can likely to be
attributed to stress due to occlusive application of the test
substance. In the dermal irritation test, the primary irritation
index (PII) was 0.33, indicating no irritant. For the ocular
irritation test, the left eye of a rabbit was applied with the test
substance, and then compared to the right eye, which was not
applied with the test substance. During the experimental period,
general symptoms and abnormal changes in body weight related to
application of the test substance were not observed. After
application of the test substance, the eye examination showed that
the index of acute ocular irritation (IAOI) was "0", indicating no
irritant. Therefore, these results indicate that the novel
bacteriophage .PHI.CJ11 has no toxicity.
Further, toxicity assay was performed by single oral administration
of Sprague-Dawley rats with .PHI.CJ11. A test
substance-administered group treated with 1.times.10.sup.11 PFU/kg
of .PHI.CJ11 and an excipient control group treated with a vehicle
[20 mM Tris-HCl (pH 7.0)+2 mM MgCl.sub.2] as an excipient were
prepared, and 10 rats of each group (5 each of female and male
sexes) were orally administered with a single dosage. Mortality,
general symptoms, changes in body weight, and autopsy findings were
monitored for 2 weeks and compared to each other. Monitoring was
conducted every 6 hours, starting from 30 minutes to 1 hour after
administration on the day of administration. Then, general symptoms
were monitored once a day for 14 days, and recorded thereof (Tables
4 and 5).
TABLE-US-00004 TABLE 4 Mortality and general symptoms after oral
administration of .PHI.CJ11 Done Day after treatment Sex (pfu) 1 2
3 4 5 6 7 8 9 10 11 12 13 14 Mortality Male Control 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 10.sup.11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female
Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10.sup.11 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0
TABLE-US-00005 TABLE 5 Autopsy findings after oral administration
of .PHI.CJ11 Done Sex (PFU) gross finding Frequency.sup.A Male
Control No gross 5/5 finding 10.sup.11 No gross 5/5 finding Female
Control No gross 5/5 finding 10.sup.11 No gross 5/5 finding
.sup.ANumber of animal with the sign/number of animals
examined.
As shown in Tables 4 and 5, none of them died, and neither toxic
symptoms nor noticeable clinical symptoms were generated by
.PHI.CJ11. The results are summarized in Tables 4 and 5. Body
weights were recorded before administration and 1, 3, 7, 10 and 14
days after administration. No significant changes were observed in
body weight compared to the control group.
Meanwhile, the results of body weight changes indicate that
.PHI.CJ11 does not cause a toxic reaction sufficient to reduce
appetite or to change body weight. These results are shown in FIG.
9. FIG. 9 is the results of body weight changes due to toxicity
after single oral administration of Sprague-Dawley rats with
.PHI.CJ11. As shown in FIG. 9, observation of body weight changes
before administration and 1, 3, 7, 10 and 14 days after
administration with .PHI.CJ11 showed that no significant changes in
body weight were found in comparison with the control group
(.box-solid.; male control, .quadrature.; .PHI.CJ11 male,
.circle-solid.; female control, .smallcircle.; .PHI.CJ11
female).
Therefore, it was found that ADL of the novel bacteriophage
.PHI.CJ11 exceeds 1.times.10.sup.11 PFU/kg in both female and male
rats, and thus it is non-toxic.
SEQUENCE LISTINGS
1
1411643DNAbacteriophage CJ11 1taaactgccg gcttaatatt aaggggagga
gcccattatg agtaaatatg gttttgtaaa 60taacggactt aatcaaagta tgggctccca
gcagcccatg ccgggtctgg ataaccccta 120cttacaacag ttgcaacagc
gtttagcaga agcgcaacag atgcagcaac aactccagca 180aaatcctgga
agtgttagtc ctatgcaaat gatgcagcaa atgaatgggc agtcgcaaat
240gccccaacaa atgcagcaac agatgccgca gcagcaagtt cagcaacaaa
cccaacaacc 300gcaggtttct gcggaaggac aggcagtact agcccttttc
gaagacttcg caaagacaga 360ggatgggaag cagcttgtat cacttatggg
taagtttaat agcttctgcc aaagccaagt 420tgcaaaagct caaaatggtg
gtaataactc ctaaggagga attatgtgtt gcagaaaatc 480tgtttcgtgc
tgcccaatgc ctgtagcacg ttgttgctct tcagcaatgt ttacacaaat
540accggcgttc aatccgtttg cctttaagcc tactcttatc ctgcccccta
gagtagattt 600tcagtcaagg ttggcctccc gtatgggtgg ttgctgtaat
aagagtatat ggttctaaag 660aaccaaacaa taaagcccct agtcgaaaga
cttaggggct tttcttttat cttaaataca 720gaaaagccct gcgctctcca
gggaacgcag ggcttacttc agatgttacg tgcggatatt 780atttattacg
catatccatg atcatttggc catcatagcc agtgccaacc acagtctgcg
840gtacaccacc tttggtattt ctgagcacga atcatttcaa ctttccagtt
gcttccagcg 900aatcatctca ggagtaatgg tacgttgcaa tgcggcgtta
gcttcagctt ctttcttagc 960tgcgtacagt ttggcatctg cgtcacgttc
gttagcaata gcttggttat tacgagcttc 1020gcgatctgct tctgcttgtt
taaccttctg ctgtgcttct tgttcaacac gagccagttc 1080agctttcgca
gcattaacct gttcttcacg aactttggta ttctgtacct gttccatgat
1140taccggtggc aaagtaatat cctgaagaaa tacctgctta actgtgtaac
catatgggcg 1200tgcatactct tcaacttcct gttgaattgc agtttgcaat
tgagcctgaa ttttagcatc 1260aaacaaatct tgtgctttag gtacagactt
accaaactca cgaatagtag acagtaattt 1320ttcagttaca tatttgtcta
acgcttgatc ctgagtacct gcattaatac ggttaatcgg 1380tgccttagaa
ccatcaaact gcaacataac agtcaggtca acagtggatt taaacttatc
1440ctgactagga acctgaagtt tatctaattt tagagcaata tctttagtac
taaaagtatc 1500gaaagaagca aaaggattta caatatgaaa gccgggtaat
actgggctag ggtcaacttt 1560acccaggaaa gtctgggttt taacagtacc
gtcttgaaca acagtatagg agttaagggc 1620cagaactaaa cctaccaaac caa
164321464DNAbacteriophage CJ11 2taggtataca ctgggatact aactcttcta
aatggaaggt agaaataact aactgtatag 60gtgttaaaat atatggtggg ttattccctt
atgaagaatt agagttagct ataaataaag 120ctaacgaact tagagcacag
catctaggta tggattccag gcaagaacta ttcaaaggat 180atatttgccg
acgtgaggac ttagataagt aaaattgcat ttggcaatag ctcataaatg
240atgtataata tatacataaa tttgagagag aaagtttcgg attgataaga
aagtccgaag 300cagaaaaata aaaattttag ttgctaaatt ctctcgaaat
ctagtataat atatacataa 360attcgaggag aaaacaaaaa ttaaattctt
cgattatgaa aagctatact tactagctag 420aggaaattcc gacctaatta
ttaagttatt caaaagaatg cttacagagc ctgatgctca 480ccaattattg
gtcggttcct cattcatttt gaatgaatca acaatagttg ataatccaaa
540taaattgtct aatagacaac tggcagaata tctaggaatt ttaagtctac
gaaattatgc 600cgaatacaag tttacaaacg atcctagttt ggacatacaa
tatgttccag tatggatacc 660acgtttagta atcgacacta acccactaat
cgcaattaac aaatcgaaat taatctttaa 720agaggaaata aaatatggct
aagtcttggg gcgaaactac tggcggttct aacgataaaa 780tcgaattcct
gaagttcaac aacggtatca ctcgtgttcg tatcgtttct ggtgttcttc
840cacgttatgt ctattggctg actaataaag agggtagcgt agctcctttc
gaatgtctcc 900gttttaaccg tgacaaagag agctttgttc gtggtaaagc
tgatccggtt catgagatgg 960gcttcttcga gaaagaactg gataaagatg
gtaatcgtgt tccgctgaaa ccgaagaaaa 1020actatatcgc ttttgttatc
gaccgttctg ataacaaact gaaagtaatg gaagtcaagg 1080ctactattct
gaaaggcatc cagtctatca tgaagcagtt gagtctggca actccgtttg
1140atattgatat ttctatcgag aaaaaaggta aaggtttcga tactgagtat
gatgtacagc 1200agattgctgc tatgcagttc cagattaagc tgcaagatcc
taacagtgca gagtctaagc 1260aatatgctgc aggcatgcaa gcttggcgta
atcatggtca tagctgtttc ctgtgtgaaa 1320ttgttatccg ctcacaattc
cacacaacat acgagccgga agcataaagt gtaaagcctg 1380gggtgcctaa
tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
1440gtcgggaaac ctgtcgtgcc agct 146431324DNAbacteriophage CJ11
3tactgaatct atataagaaa caacaaattc gcgaacgtta gcgccagctc ctctatccca
60ttcccattgt acacggagat catatctttc tttaccatca gcaatccttg cagctttgaa
120agtaatgttg ttaggagcag taggaggtac aaagttatac tctacagtaa
gaactttgga 180aaattcaaag tatccagaag agtctacagt tacaccatct
ggcatggtaa cttgcccaga 240tatgcgtact ttgtaatccc caacaggaac
accaccaaat ctaatagtgg gagatagtgg 300acctatataa tactttaccc
acgggctatt tgaaggttga gtacttttta actcaatagt 360acaataacta
gcttctcctg tagtctctat aactactata ggggcaccta cacccacatc
420tactggatca gcctctgatc tagcggcagt aatagtaggt tttctcttag
tactaaaggt 480agttttattg gataagttta tgccaatttt agcctctaat
agctcagagt ctataataga 540gtcatagaaa gccccttgaa cctcataaga
agttgaaggg gttagattat taatcattac 600aaagaaagta tctataccgg
tatagtcacg tctatcaata ctttctccta actttaacca 660aaaggatcta
ccaataacgt cataatcagt atatatagag tgctggacat atgccattgt
720atatccagtc attatactat ttaagaccat tttggctggt gcattattcg
aaatcattaa 780atggatgccc actccacaga ctgttcccca tcatctcctt
ctgctctaat acgaaaatat 840agttttctat tgatccctag agcatcactg
ttatggagag caaaatctgc cttattatac 900attaataaat agtcataagt
atactgattt tcaatacgta cacttcttag cattctattc 960tgggaatcat
agatctccag agtatagaat atactttcta ttatatcttc ttctggtatc
1020ctatcccaag ctagttttac gtctgggccg acaaactcag ttacatctcc
agaagccgta 1080ttagttaccc taaaattaga tactacgctt aggtttttag
cagagttaag ttctatagat 1140aatgttactg gagaacttct tctaccgttt
atatccacag cccttatttc aaatattgct 1200aaacctgcag gttctccaac
tatctcttgg atcatgcgtt cattgggatt tgtttctagt 1260tgctgcacaa
tatacggctc agcatggcct gaatgtacga tagaatagta aactacgtta 1320ttag
132441666DNAbacteriophage CJ11 4cagaaaataa cattaccttc tttatctgtt
tttagtacaa ccgtttctgg tttagtagtt 60ttaatctgat aagttgatgt actagtatca
atacctacca tattaggatc ggtaaaatta 120tttgcttcct gaacttccaa
atccgccttc cccacgatct gtctccccta gttcgtcaac 180gatttcaaaa
ttatgagttg agtagtgtgg tactactact agctgacaaa gtctttcaaa
240attttccaga gtttgaattt cagaaccata gttataaagg ttcatcttaa
tagttccacg 300atagtctgag tcaatcactc ctgcggtgtt tgcaatcatc
agatgacgct tacctaagga 360gctacgagga actaccaaac cgaaccaacc
tcgcggaatt tctaccgcga caccggtgtc 420aatcattaag gatttgcctg
gtgcaatagc acgtaaatct gcagcagggt tagtaccaaa 480gaacgctcgc
agatccatac ctgcggcatc ttcggaacca atcttaggcg tacaatctgg
540atgagttaat ttaattttaa tcattgttct gcaatctcca aaatatcttt
tgtaaattta 600tctaatacat cttgacctac agcagcaata gcatccacac
agtaggtagg taaatcaacc 660agaattaggt ttcgataaag caattcttcc
gaagcattta aattctgtat atatttctgt 720tttccaggca gtggaagctg
atcaataata tccagaacgt taccaaattc acgaataata 780ttatacccac
gctttgcccc aataccctca acacctcgga tattatcccc taaatcaccc
840ataattgctt tcagggagat aaactgctct acatcgtcaa cattatgatg
ctcatacata 900tcacgaagat gatattcacg acgtgttgtg aaagagaagc
gagatacttt atcagttaat 960aaagtatccc agtcaccatc ggtagaaatt
agccaaacat gatcatatag atgcccaatc 1020agcttaacaa tataagctgc
catatcatct gcttctaccc cacgaatagt gaaagttggg 1080aatgtagttt
cacataattc gaaagcatct ttcaagtact cgaagaattg ttcatctaat
1140gctttctcct cttccgtacg ctgcgagtat ttctcatctc gattcccttt
atactcaggg 1200agatgctcta ggcggaatgc agacttccct ttatctccta
aaactattgt agttctagca 1260gagtaagatt ttgcaagaga ctgaatagtg
gaaacataac ttgaggcaaa tggtttttta 1320ctattgttat gcttgaagcg
aaagcctaag ttagttccat cgacaatcat taggttacga 1380cgggaagcca
tttcggcttc ctcttcctca ataaattttc cccaggattt actcattatt
1440taattaagtc ctcaacagat gcatgatgta gccacggctc aaataaacca
attacgattt 1500ccatgtcttt cttatttaac accatatggg tacgactcat
taagttgtca accatcgggt 1560ctgagctatc caaagctatt aaccactgtc
ctctgtcttt tttgaatatt aatgcaggtt 1620tggagttcat ctgctcacct
tcacgtgagc actgctgcca ccactt 1666520DNAArtificial Sequenceprimer
5cccattatga tgaaataggg 20619DNAArtificial Sequenceprimer
6ctatactgtt gttcaagac 19720DNAArtificial Sequenceprimer 7gatactaact
cttctaaatg 20820DNAArtificial Sequenceprimer 8cttagactct gaactgttag
20922DNAArtificial Sequenceprimer 9ctgaatctat ataagaaaca ac
221020DNAArtificial Sequenceprimer 10cgaagtttac tattctatcg
201120DNAArtificial Sequenceprimer 11gtttttagta caaccgtttc
201219DNAArtificial Sequenceprimer 12cagatgaact ccaaacctg
191320DNAArtificial Sequenceprimer 13cgggcctctt cgctattacg
201422DNAArtificial Sequenceprimer 14aggcttaccc gtcttactgt cg
22
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